Hi,
I extracted DNA from blood samples by organic method with freshly prepared solutions.
Dissolved DNA in double distiled deionized water (Like I did for my previous samples). see figure 1 for Gel. Nanodrop concentration was good with 260/280 and 260/230 ratio above 1.8 and 2.0 respectively.
But these samples are not amplified by PCR. I run these samples with previously extracted samples. Old samples are giving very good amplification but newly extracted samples are not amplified. Figure 2.
I even tried different dilutions but No amplification in any dilution.
Then I again washed these DNA samples with isopropanol and 70% ethanol. This time dissolved DNA in elution buffer provided with Vivantis DNA extraction kit. figure 3.
again NO PCR. figure 4
I am unable to point the issue. please help in resolving the issue.
Hi
there is many reasons.
Maybe do test again is better option.
In pic 3 that you sent, your old samples are sharper and maybe had more DNA as new one, and it be caused an error in your PCR.
Keep looking.
thank you Alireza Mordadi for your answer.
Old samples are sharper but they are amplifying perfectly while new samples are not giving any amplification for same primers.
hi, dear Misbah Hussain,
your DNA sample concentration is high and another hand your DNA sample has the smear, I suggest that reduce your PCR DNA concentration.
you can use serial dilution to set up your PCR.
HI Dear Misbah Hussain,
1. If you remeber the concentration of the old sample, try to make as close as possibel for the new one(concentrating if low or diluting if high),
2. May be the pcr worked but the gell buffers, try to use a fresh buffers.
3.The old ones are on difffernt gell, you should have put in the new pcr as a positive control, to make sure that the problem is with samples but not pcr.
Good Luck!
There are many things you can try, and the ratios on the nanodrop are merely an estimation. You would have to provide more information on the type of samples (are they different species? etc) and what primers you are using (how specific are they? etc) in order to give the best advice.
For now I would say, follow the advice of the other people here and some other things you can try:
- diluting and concentrating your samples
- try a gradient for the annealing temperature
- make a series with different amounts of magnesium
- add BSA or TBT-PAR or DMSO
- change the amount of Taq you put in
I attach here a paper where they show the effects of changing the different parameters for PCR's which has helped me understand things a lot.
Good luck! PCR's can be unpredictable at times...
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